Mature Myotubes Research Articles

The increase of pericyte population in human neuromuscular disorders supports their role in muscle regeneration in vivo

Pericytes are periendothelial cells that have been involved in many different functions including a possible role as mesodermal stem/progenitor cells. In the present study we demonstrate that alkaline phosphatase (AP) expression is specific for human muscular pericytes and can be used as a marker to identify them in skeletal muscle biopsies. We studied the pericyte population in skeletal muscle biopsies from controls, myopathic and neuropathic patients. We observed a significant increase in the number of pericytes only in myopathies that correlated with the number of NCAM(+) fibres, suggesting that an active muscular degenerative/regenerative process is related to an increase in the pericyte population. AP(+) pericytes sorted from skeletal muscle samples were able to activate the myogenic programme and fuse with both mononucleate satellite cells and mature multinucleated myotubes in vitro, demonstrating that they could participate in muscle regeneration. In accordance, pericytes expressing the myogenic transcription factor MyoD were found in biopsies of myopathic biopsies. All these data support the hypothesis that, apart from satellite cells, pericytes may play an important role in muscle regeneration in adult human muscles in vivo.

ISOLATION AND SEQUENCING OF MRF4 GENE (EXON 1 THROUGH 4) FROM EGYPTIAN BUFFALO

Muscle regulatory factor 4(MRF4, also known as herculin and MYF6) is a member of MyoD family, which is involved in the differentiation and maturation of myotubes and highly expressed postnatally.The sequences of this gene are well known in cattle and small ruminants but in buffalo no available sequence was previously recorded before this research. Therefore, the main aim of this study is to isolate and sequence exon 1 (E1) through exon 4 (E4) portion of this gene, which is the most appropriate portion of MRF4 that contains single nucleotide polymorphisms (SNPs), in buffalo. To achieve this, RT-PCR was performed in which gene-specific primers flanking to E1-E4 of MRF4 were utilized in addition to PCR master mix. A PCR product of 693bp, of MRF4 gene corresponding to the expected theoretical product size, was successfully amplified. Sequencing of the purified PCR product and its alignment against a previously known bovine gene identified 4 synonymous novel SNPs in buffalo MRF4. This result indicates that the sequence of MRF4 is highly conserved between cattle and buffalo. This is a preliminary study that provides the researchers withraw data which could be used as a basis for further studies to associate these SNPs with meat quality traits in buffalo.

Comparative expression profiling identifies differential roles for Myogenin and p38α MAPK signaling in myogenesis

Skeletal muscle differentiation is mediated by a complex gene expression program requiring both the muscle-specific transcription factor Myogenin (Myog) and p38α MAPK (p38α) signaling. However, the relative contribution of Myog and p38α to the formation of mature myotubes remains unknown. Here, we have uncoupled the activity of Myog from that of p38α to gain insight into the individual roles of these proteins in myogenesis. Comparative expression profiling confirmed that Myog activates the expression of genes involved in muscle function. Furthermore, we found that in the absence of p38α signaling, Myog expression leads to the down-regulation of genes involved in cell cycle progression. Consistent with this, the expression of Myog is sufficient to induce cell cycle exit. Interestingly, p38α-defective, Myog-expressing myoblasts fail to form multinucleated myotubes, suggesting an important role for p38α in cell fusion. Through the analysis of p38α up-regulated genes, the tetraspanin CD53 was identified as a candidate fusion protein, a role confirmed both ex vivo in primary myoblasts, and in vivo during myofiber regeneration in mice. Thus, our study has revealed an unexpected role for Myog in mediating cell cycle exit and has identified an essential role for p38α in cell fusion through the up-regulation of CD53.

Did giant mitochondria delay muscle maturation? An uncommon congenital myopathy

Mitochondria are semi-autonomous organelles that are able to change their shape, size, location, and number inside the living cell. Mitochondrial division is an extremely important process, because cell survival depends on there being an adequate number of mitochondria in each cell. The dynamics of these organelles are particularly important for normal embryonic cell development. We studied morphological and ultrastructural features of muscle-cell immaturity, with the appearance of abnormal giant mitochondria, in a child with an unusual congenital myopathy. The biceps brachii and quadriceps femoris muscles were analyzed at the light and electron microscopy levels. Ultrastructural features of muscle-cell immaturity were manifested in the presence of primary and mature myotubes coexisting with abnormal giant mitochondria. Failure of mitochondrial fusion/fission machinery at an early stage of development may lead to delayed muscle-cell maturation.

Multiple‐staged Regulation of Myogenic Differentiation by Prostaglandin E2

We showed that conditioned media from osteocytes accelerates myogenesis in C2C12 myoblasts, which suggests signaling from bone to muscle cells. Since osteocytes secrete Prostaglandin E2 (PGE2), one of the major PGs synthesized from arachidonic acid through cyclooxygenase pathways, we started a systematic investigation of PGE2 roles in myogenesis. C2C12 myoblasts were used to investigate the effect and related molecular mechanisms of PGE2 on myogenic differentiation. Expression of PGE2 receptors (EP1‐EP4) was determined by real‐time PCR. EP1 and EP4 were highly expressed, whereas EP2 and EP3 were not detectable. Specific inhibitors SC 51322 for EP1 or L161,982 for EP4 significantly attenuated myogenesis. EP1 inhibition reduced the number of early myotubes, and subsequently affected the development of mature myotubes. On the other hand, EP4 inhibition did not affect the generation of early myotubes, but the fusion of early myotubes was dramatically inhibited. Interestingly, expression of two key myogenic regulatory factors, MyoD and Myogenin, was downregulated by EP1, but not by EP4 inhibition. These data provide evidence for the regulation of myogenesis by PGE2 via EP1 and EP4. The different effects of EP1 and EP4 inhibition suggest PGE2 signaling in myogenic differentiation is complex and multiple‐faceted. Support: NIH RC2 AR058962, Missouri Life Sciences Research Board(MB), and NIDCR DE021888(OJI).

Chemokine-like receptor 1 regulates skeletal muscle cell myogenesis

The chemokine-like receptor-1 (CMKLR1) is a G protein-coupled receptor that is activated by chemerin, a secreted plasma leukocyte attractant and adipokine. Previous studies identified that CMKLR1 is expressed in skeletal muscle in a stage-specific fashion during embryogenesis and in adult mice; however, its function in skeletal muscle remains unclear. Based on the established function of CMKLR1 in cell migration and differentiation, we investigated the hypothesis that CMKLR1 regulates the differentiation of myoblasts into myotubes. In C(2)C(12) mouse myoblasts, CMKLR1 expression increased threefold with differentiation into multinucleated myotubes. Decreasing CMKLR1 expression by adenoviral-delivered small-hairpin RNA (shRNA) impaired the differentiation of C(2)C(12) myoblasts into mature myotubes and reduced the mRNA expression of myogenic regulatory factors myogenin and MyoD while increasing Myf5 and Mrf4. At embryonic day 12.5 (E12.5), CMKLR1 knockout (CMKLR1(-/-)) mice appeared developmentally delayed and displayed significantly lower wet weights and a considerably diminished myotomal component of somites as revealed by immunolocalization of myosin heavy chain protein compared with wild-type (CMKLR1(+/+)) mouse embryos. These changes were associated with increased Myf5 and decreased MyoD protein expression in the somites of E12.5 CMKLR1(-/-) mouse embryos. Adult male CMKLR1(-/-) mice had significantly reduced bone-free lean mass and weighed less than the CMKLR1(+/+) mice. We conclude that CMKLR1 is essential for myogenic differentiation of C(2)C(12) cells in vitro, and the CMKLR1 null mice have a subtle skeletal muscle deficit beginning from embryonic life that persists during postnatal life.

PED/PEA-15 induces autophagy and mediates TGF-beta1 effect on muscle cell differentiation

TGF-beta1 has been shown to induce autophagy in certain cells but whether and how this action is exerted in muscle and whether this activity relates to TGF-beta1 control of muscle cell differentiation remains unknown. Here, we show that expression of the autophagy-promoting protein phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15) progressively declines during L6 and C2C12 skeletal muscle cell differentiation. PED/PEA-15 underwent rapid induction upon TGF-beta1 exposure of L6 and C2C12 myoblasts, accompanied by impaired differentiation into mature myotubes. TGF-beta1 also induced autophagy in the L6 and C2C12 cells through a PP2A/FoxO1-mediated mechanism. Both the TGF-beta1 effect on differentiation and that on autophagy were blocked by specific PED/PEA-15 ShRNAs. Myoblasts stably overexpressing PED/PEA-15 did not differentiate and showed markedly enhanced autophagy. In these same cells, the autophagy inhibitor 3-methyladenine rescued TGF-beta1 effect on both autophagy and myogenesis, indicating that PED/PEA-15 mediates TGF-beta1 effects in muscle. Muscles from transgenic mice overexpressing PED/PEA-15 featured a significant number of atrophic fibers, accompanied by increased light chain 3 (LC3)II to LC3I ratio and reduced PP2A/FoxO1 phosphorylation. Interestingly, these mice showed significantly impaired locomotor activity compared with their non-transgenic littermates. TGF-beta1 causes transcriptional upregulation of the autophagy-promoting gene PED/PEA-15, which in turn is capable to induce atrophic responses in skeletal muscle in vivo.

HUWE1 ubiquitinates MyoD and targets it for proteasomal degradation

MyoD is a tissue-specific transcriptional activator that acts as a master switch for muscle development. It activates a broad array of muscle-specific genes, which leads to conversion of proliferating myoblasts into mature myotubes. The ubiquitin proteasome system (UPS) plays an important role in controlling MyoD. Both its N-terminal residue and internal lysines can be targeted by ubiquitin, and both modifications appear to direct it for proteasomal degradation. The protein is short-lived and has a half-life of ∼45min in different cells. It was reported that MyoD can be ubiquitinated by MAFbx/AT-1, but accumulating lines of experimental evidence showed that other ligase(s) may also participate in its targeting. Here we describe the involvement of HUWE1 in the ubiquitination and proteasomal degradation of MyoD. Furthermore, we show that the ligase can ubiquitinate the protein in its N-terminal residue.

Protein Tyrosine Phosphatase-Like A Regulates Myoblast Proliferation and Differentiation through MyoG and the Cell Cycling Signaling Pathway

Protein tyrosine phosphatase-like A (PTPLa) has been implicated in skeletal myogenesis and cardiogenesis. Mutations in PTPLa correlated with arrhythmogenic right ventricular dysplasia in humans and congenital centronuclear myopathy with severe hypotonia in dogs. The molecular mechanisms of PTPLa in myogenesis are unknown. In this report, we demonstrate that PTPLa is required for myoblast growth and differentiation. The cells lacking PTPLa remained immature and failed to differentiate into mature myotubes. The repressed MyoG expression was responsible for the impaired myoblast differentiation. Meanwhile, impeded cell growth, with an obvious S-phase arrest and compromised G(2)/M transition, was observed in PTPLa-deficient myoblasts. Further study demonstrated that the upregulation of cyclin D1 and cyclin E2 complexes, along with a compromised G(2)/M transition due to the decreased CDK1 (cyclin-dependent kinase 1) activity and upregulated p21, contributed to the mutant cell S-phase arrest and eventually led to the retarded cell growth. Finally, the transcriptional regulation of the PTPLa gene was explored. We identified PTPLa as a new target gene of the serum response factor (SRF). Skeletal- and cardiac-muscle-specific SRF knockouts resulted in significant decreases in PTPLa expression, suggesting a conserved transcriptional regulation of the PTPLa gene in mice.

Modulation of alignment and differentiation of skeletal myoblasts by biomimetic materials

Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. We developed a novel cell culture technique that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. Alignment and fusion of myoblasts into parallel arrays of multinucleated myotubes are critical in skeletal muscle tissue engineering and more micro-patterning techniques and surface engineering have been tested by switching differentiating myotubes from growth medium (GM) to differentiative media (DM). One of the goals of tissue engineering is to develop tools allowing in vitro construction and mimicking of the final tissue architectures. The fabrication of polyelectrolyte multilayers (PEMs) may represent a promising approach for recreating physiological nanometer-sized cell environments in vitro. In this study we describe a method for generating biomimetic microstructured surfaces that promote cell adhesion and differentiation of parallel arrays of mature C2C12 myotubes continuously maintained in GM for 7 days. The structure consists of a 'double-sheet' PDMS structure that provides 'compliant' or 'stiff' microdomains to guide the cell self-patterning, coupled to layer-by-layer (LbL) self-assembled multilayers of biocompatible polyelectrolytes that promote C2C12 myoblasts alignment and differentiation. Our findings have relevance to the interpretation of in vitro data as well as to the study of cellular interactions with biomaterials.

Abstract P113: Protein Tyrosine Phosphatase-Like A Is a Unique Regulator for Myogenesis

Background: Protein tyrosine phosphatase-like A (PTPLa) is highly expressed in skeletal and cardiac muscle. Therefore, it has been implicated to play a role in skeletal myogenesis and cardiogenesis. Mutations in PTPLa correlated with arrhythmogenic right ventricular dysplasia in humans and congenital centronuclear myopathy with severe hypotonia in dogs. However, the molecular mechanisms of PTPLa in myogenesis are unknown. Method and results: Mouse myoblast C2C12 cells were used to generate PTPLa deficient and PTPLa overexpression cell lines and the cells were cultured in proliferating and differentiating media, respectively, to study regulatory role of PTPLa in cell proliferation and differentiation. We demonstrated that PTPLa was required for myoblast growth and differentiation. The cells lacking PTPLa remained immature and failed to differentiate into mature myotubes along with repressed MyoG expression. To define the role of MyoG in PTPLa-mediated myoblast differentiation, myoblast cell line lacking MyoG expression was generated. Two cell lines, the PTPLa-deficient and MyoG-deficient myoblasts were used to demonstrate that PTPLa-mediated myoblast differentiation was through MyoG regulation. Meanwhile, the impeded cell growth with an obvious S-phase arrest was observed in PTPLa-deficient myoblasts. Further study demonstrated that the upregulation of cyclin D1 and cyclin E2 complexes along with a compromised Cdk1 activity contributed to the mutant cell S-phase arrest and eventually led to the retarded cell growth. Finally, the transcriptional regulation of PTPLa gene was explored. We identified that PTPLa was a new target gene of serum response factor (SRF). Skeletal- and cardiac-specific SRF knockouts resulted in significant decreases in PTPLa expression, suggesting a conserved transcriptional regulation of PTPLa gene in mice. Conclusion: PTPLa is a unique pro-myogenic factor regulated by SRF. It regulates myoblast proliferation and differentiation through MyoG and cell cycling signaling pathway.

Optically controlled contraction of photosensitive skeletal muscle cells

As the skeletal muscle cell is an efficient force transducer, it has been incorporated in bio-microdevices using electrical field stimulation for generating contractile patterns. To improve both the spatial and temporal resolutions, we made photosensitive skeletal muscle cells from murine C2C12 myoblasts, which express channelrhodopsin-2 (ChR2), one of archaea-type rhodopsins derived from green algae Chlamydomonas reinhardtii. The cloned ChR2-expressing C2C12 myoblasts were made and fused with untransfected C2C12 to form multinucleated myotubes. The maturation of myotubes was facilitated by electrical field stimulation. Blue LED light pulse depolarized the membrane potential of a ChR2-expressing myotube and eventually evoked an action potential. It also induced a twitch-like contraction in a concurrent manner. A contraction pattern was thus made with a given pattern of LED pulses. This technique would have many applications in the bioengineering field, such as wireless drive of muscle-powered actuators/microdevices.

Development of an in vitro model of myotube ischemia

Critical limb ischemia causes severe damage to the skeletal muscle. This study develops a reproducible model of myotube ischemia by simulating, in vitro, the critical parameters that occur in skeletal muscle ischemia. Monolayers of C2C12 myoblasts were differentiated into mature myotubes and exposed to nutrition depletion, hypoxia and hypercapnia for variable time periods. A range of culture media and gas mixture combinations were used to obtain an optimum ischemic environment. Nuclear staining, cleaved caspase-3 and lactate dehydrogenase (LDH) release assay were used to assess apoptosis and myotube survival. HIF-1α concentration of cell lysates, pH of conditioned media as well as partial pressures of oxygen (PO2) and carbon dioxide (PCO2) in the media were used to confirm ischemic simulation. Culturing myotubes in depleted media, in a gas mixture containing 20% CO2+80% N2 for 6–12 h increased the PCO2 and decreased the pH and PO2 of culture media. This attempts to mimic the in vivo ischemic state of skeletal muscle. These conditions were used to study the potential tissue-protective effects of erythropoietin (EPO) in C2C12 myotubes exposed to ischemia. EPO (60 ng/ml) suppressed LDH release, decreased cleaved caspase-3 and reduced the number of apoptotic nuclei, suggesting significantly decreased ischemia-induced apoptosis in myotubes (P<0.01) and a potential role in tissue protection. Additional therapeutic agents designed for tissue protection can also be evaluated using this model.

Expression of miR-1, miR-133a, miR-133b and miR-206 increases during development of human skeletal muscle

BackgroundMicroRNAs (miRNAs) are small RNA molecules that post-transcriptionally regulate gene expression and have been shown to play an important role during development. miR-1, miR-133a, miR-133b and miR-206 are expressed in muscle tissue and induced during muscle cell differentiation, a process that directs myoblasts to differentiate into mature myotubes, which are organized into myofibers. Although miR-1, miR-133a, miR-133b and miR-206 are well-studied in muscle, there is no information about their expression and function during human development. The purpose of this study was to determine the profile of these miRNAs in muscle cells isolated from different stages of human development.ResultsWe examined the levels of miR-1, miR-133a, miR-133b and miR-206 during the development of human foetus. All four miRNA levels were found increased during late stages of human foetal muscle development. Increases in the expression levels of these miRNAs were proportional to the capacity of myoblasts to form myotubes. Changes in miRNA levels during human foetal development were accompanied by endogenous alterations in their known targets and also in their inducer, MyoD. Ectopic MyoD expression caused an induction of muscle cell differentiation in vitro, accompanied by an increase in the levels of miR-1, miR-133a, miR-133b and miR-206.ConclusionsThis study provides data about the profile of four miRNAs in human muscle cells isolated during different stages of foetal development. These results may shed light on the differentiation of muscle cells and regulation of muscle formation through miRNAs, during the development of human foetus.

In vitro expression profiling of myostatin, follistatin, decorin and muscle-specific transcription factors in adult caprine contractile myotubes

Skeletal muscle is one of the several adult postmitotic tissues that retain the capacity to regenerate, which relies on a population of quiescent precursors, termed satellite cells. Proliferation and differentiation of myoblasts to form mature myotubes in vitro has been a valuable tool in the characterization of the cellular events during myogenesis, which is a multistep process starting with progenitor cell proliferation, followed by their exit from the cell cycle, differentiation, alignment, and fusion to form multinucleated myotubes. A typical feature during muscle differentiation is the variation in expression of various genes along with myogenic factors. In this experiment, mRNA level of myostatin, follistatin, decorin and three muscle-specific transcription factors in adult caprine contractile myotubes have been studied through quantitative real time PCR. We observed that the expression level of myostatin, decorin, Myf5 and myogenin transcripts were significantly higher in contractile myotubes compared to myoblast monolayer (P < 0.05), and follistatin level was similar in both types of cells, whereas MyoD transcript level was significantly high in monolayer culture which might be due heterogeneity of myoblast population. It is concluded that the information generated would provide the base line information as well as monitoring markers to undertake experiments aimed at modulating muscle growth.

Isolation and transcriptome analysis of adult zebrafish cells enriched for skeletal muscle progenitors

Introduction: Over the past 10 years, the use of zebrafish for scientific research in the area of muscle development has increased dramatically. Although several protocols exist for the isolation of adult myoblast progenitors from larger fish, no standardized protocol exists for the isolation of myogenic progenitors from adult zebrafish muscle. Methods: Using a variant of a mammalian myoblast isolation protocol, zebrafish muscle progenitors have been isolated from the total dorsal myotome. These zebrafish myoblast progenitors can be cultured for several passages and then differentiated into multinucleated, mature myotubes. Results: Transcriptome analysis of these cells during myogenic differentiation revealed a strong downregulation of pluripotency genes, while, conversely, showing an upregulation of myogenic signaling and structural genes. Conclusions: Together these studies provide a simple, yet detailed method for the isolation and culture of myogenic progenitors from adult zebrafish, while further promoting their therapeutic potential for the study of muscle disease and drug screening. Muscle Nerve 43: 741–750, 2011

Skeletal Muscle 11beta-HSD1 Controls Glucocorticoid-Induced Proteolysis and Expression of E3 Ubiquitin Ligases Atrogin-1 and MuRF-1

Recent studies demonstrated expression and activity of the intracellular cortisone-cortisol shuttle 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in skeletal muscle and inhibition of 11beta-HSD1 in muscle cells improved insulin sensitivity. Glucocorticoids induce muscle atrophy via increased expression of the E3 ubiquitin ligases Atrogin-1 (Muscle Atrophy F-box (MAFbx)) and MuRF-1 (Muscle RING-Finger-1). We hypothesized that 11beta-HSD1 controls glucocorticoid-induced expression of atrophy E3 ubiquitin ligases in skeletal muscle. Primary human myoblasts were generated from healthy volunteers. 11beta-HSD1-dependent protein degradation was analyzed by [3H]-tyrosine release assay. RT-PCR was used to determine mRNA expression of E3 ubiquitin ligases and 11beta-HSD1 activity was measured by conversion of radioactively labeled [3H]-cortisone to [3H]-cortisol separated by thin-layer chromatography. We here demonstrate that 11beta-HSD1 is expressed and biologically active in interconverting cortisone to active cortisol in murine skeletal muscle cells (C2C12) as well as in primary human myotubes. 11beta-HSD1 expression increased during differentiation from myoblasts to mature myotubes (p<0.01), suggesting a role of 11beta-HSD1 in skeletal muscle growth and differentiation. Treatment with cortisone increased protein degradation by about 20% (p<0.001), which was paralleled by an elevation of Atrogin-1 and MuRF-1 mRNA expression (p<0.01, respectively). Notably, pre-treatment with the 11beta-HSD1 inhibitor carbenoxolone (Cbx) completely abolished the effect of cortisone on protein degradation as well as on Atrogin-1 and MuRF-1 expression. In summary, our data suggest that 11beta-HSD1 controls glucocorticoid-induced protein degradation in human and murine skeletal muscle via regulation of the E3 ubiquitin ligases Atrogin-1 and MuRF-1.

Patterning the differentiation of C2C12 skeletal myoblasts

Mammalian cells are sensitive to the physical properties of their micro-environment such as the stiffness and geometry of the substrate. It is known that the stiffness of the substrate plays a key role in the process of mammalian myogenesis. However, the effect of geometrical constraints on the process of myogenic differentiation needs to be explored further. Here, we show that the geometrical cues of substrates can significantly influence the differentiation process of C2C12 skeletal myoblasts. Three different geometries including lines of different widths, tori of different inner diameters, and hybrid structures (linear and circular features with different arc degrees) were created by micro-contact printing of fibronectin on the surface of Petri dishes. The differentiation of C2C12 cells was studied over a period of seven days and was quantified; we report the differentiation parameters of (1) fusion index, (2) degree of maturation, (3) alignment, and (4) response to electrical pulse stimulation (EPS). Hybrid structures with the smallest arc degree (hybrid 30°) showed the best results for all four differentiation parameters. The hybrid 30° pattern exhibits an ~2-fold increase in the fusion index when compared to the line patterns and an ~3-fold increase when compared to the toroid patterns. The hybrid 30° also showed a higher maturation index compared to the line or the toroid patterns. In response to electrical stimulation (20 V, 50 ms pulse, 1 Hz), mature myotubes on hybrid 30° patterns showed an ~2-fold increase in cellular displacement when compared to myotubes on the line and torus patterns. We tested the influence of C2C12 cell density on fusion and maturation indices, and the results suggest that density does not exert significant influence on cellular differentiation under these conditions. Our results can have important implications in engineering skeletal muscle tissues and designing muscle cell bio-actuators.

Cutting Edge Issues in Polymyositis

Skeletal muscle is the target tissue of immunoflogistic processes in patients affected with idiopathic inflammatory myopathies (IIM). IIM are classified into three major forms: polymyositis (PM), dermatomyositis (DM), and inclusion body myositis. Recent data suggest that, in the major subsets of myositis, antigens in muscles drive a B-cell antigen-specific immune response. Moreover, some non-immunological mechanisms have been advocated. In this regard, an increased expression of Jo-1 and Mi-2 in muscle biopsies from PM and DM patients compared to normal muscle has been demonstrated; these candidate autoantigens in myositis are expressed at high levels in regenerating muscle cells rather than in mature myotubes. Myositis autoantigen upregulation has also been observed in neoplastic tissues, thus representing a potential link between cancer and autoimmunity in myositis. Myositis-specific autoantibodies (MSA) are disease markers and target intracellular proteins involved in key processes such as translocation and nuclear transcription. Myositis target antigens encompass aminoacyl-tRNA synthetases, the Mi-2 helicase/histone deacetylase protein complex, the signal recognition particle ribonucleoprotein, together with novel target antigens including p155/140, CADM-140, and SAE. Despite their high specificity for autoimmune myositis, MSA target non-muscle restricted proteins ubiquitary to all cell types, making the specific muscle involvement difficult to explain. Non-immunological mechanisms also seem to contribute to the pathogenesis of IIM; activation of endoplasmic reticulum stress response due to muscle regeneration and inflammation but independent to MHC-1 up-regulation has been recently reported in patients with myositis.

Over-expression of NYGGF4 (PID1) inhibits glucose transport in skeletal myotubes by blocking the IRS1/PI3K/AKT insulin pathway

Introduction Defects in insulin-stimulated glucose uptake in muscle are the important early events in the pathogenesis of insulin resistance. NYGGF4 (also named PID1) is a recently discovered gene which is suggested to be associated with obesity-associated insulin resistance. In this study, we aimed to investigate the effects of NYGGF4 on glucose uptake and insulin signaling in rat skeletal muscle cells. Methods Rat L6 myoblasts were transfected with either an empty vector or an NYGGF4-expressing vector and induced to differentiate into mature L6 skeletal myotubes. Glucose uptake was determined by measuring uptake of 2-deoxy- d -[ 3H] glucose. Immunoblotting was performed to detect the translocation of insulin-sensitive glucose transporter 4 (GLUT4). Immunoblotting was also used to measure phosphorylation and total protein levels of the insulin signaling proteins including insulin receptor (IR), insulin receptor substrate 1 (IRS1), Akt, extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun-N-terminal kinase (JNK). Results NYGGF4 over-expression in L6 skeletal myotubes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS1 and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, p38, or JNK. Conclusions Over-expression of NYGGF4 inhibits glucose transport in skeletal myotubes by blocking the IRS1/PI3K/AKT insulin pathway. These observations highlight the potential role of NYGGF4 in glucose homeostasis and the development of insulin resistance in obesity.